1. Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Diagnosis of latent tuberculosis infection: the role of IGRAs Delia Goletti, MD, PhD Translational Research Unit, INMI ERS online course on tuberculosis, March 8 th, 2011

2. March 8 th, 2011 Best wishes to all women in the “woman day”!

3. Agenda LTBI definition TST IGRA New experimental tests

4. Global burden of TB in 2009 Estimated number of Cases N. (%) Deaths N. (%) All forms of TB Women 9.4 million 3.3 million (35%) 1.3 million 380,000 (29%) HIV-associated TB1.1 million (12%)400,000 (36%) MDR-TB440,000 (4.6%)150,000 (34%) WHO report, 2010

5. Immunity against M. tuberculosis Adapted by Schwander and Dheda, 2011

6. Different stages of tuberculosis Infection eliminated with or without T cell priming Infection (latent tuberculosis infection, LTBI) Recent ( with half of the total risk to progress to active disease within 2 years ) Latent ( with half of the risk to progress to active disease during the whole life time )  Active disease Bacterial load ? Adapted from: Young et al, Trends in Immunol, 2009 Barry et al, Nature Reviews Microbiol, 2009

7. Latent infection with M. tuberculosis Direct identification of M. tuberculosis in individuals who are latently infected is not possible. LTBI is a status characterized by the absence of clinical, and radiological evidence of TB disease and the diagnosis is performed by an immune test that ascertains M. tuberculosis-specific immune responses (positive TST or an IGRA) due to: a presumptive infection with M. tuberculosis (Mack et al, ERJ 2009) a condition where human tissues contain living M. tuberculosis that persists in a state of altered metabolism that potentially may later reactivate (Opie and Aronson, 1927; Ulrichs et al, JID 2005)

8. Evidence for the existence of LTBI? TST + contacts have a higher risk for developing TB that is reduced by INH treatment (Ferebee et al, 1962; Veening et al, 1968; Egsmose et al, 1965) Erkens C et al. Eur Respir J 2010 Treatment regimenEfficacy/effectivenessEvidence 12 mo INH93%/75%A 9 mo INH90%C 6 mo INH69%65%A 4 mo RIFunknown (>3 mo INH/RIF)C 3 mo INH/RIFequivalent to 6 mo INHA

9. Latent infection with M. tuberculosis : size of the problem It is estimated (by TST) that 2 billion people globally are latently infected with M. tuberculosis (Sudre et al, 1992) LTBI subjects may develop active TB because of the waning of effective host immune responses due to: chronic diseases such as diabetes, alcoholic liver disease, malnutrition, immunosuppression due to : HIV co-infection steroids or other immunosuppressive drugs

10. Agenda LTBI definition TST IGRA New experimental tests

11. Principle of the Tuberculin skin test (TST) intradermal antigen-inoculation uptake by antigen-presenting cells antigen-presentation in lymph nodesinteraction with T-cells cytokine-releaseclonal T-cell expansion increase in capillary permeability influx of memory T cells into the test area palpable induration (max. at 48-72 h)

12. Limitations of the TST Reagent: Purified protein derivative (PPD) commonly shared among different Mycobacteria (M.tuberculosis, BCG and atypical mycobacteria) Variability: Reproducibility in giving the test Subjectivity in reading the test Logistics Repeat visit needed 3 days before result

13. Positive TST M. tuberculosis Active TB disease Latent TB infection Non Tuberculosis Micobacteria (NTM) Exposure to environmental mycobacteria or disease BCG-vaccination TST TST does not distinguish among all these different clinical situations

14. Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

15. Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

16. Species specificities of ESAT-6 and CFP-10 Environmental strains Antigens ESATCFP M abcessus-- M avium-- M branderi-- M celatum-- M chelonae-- M fortuitum-- M gordonii-- M intracellulare-- M kansasii++ M malmoense-- M marinum++ M oenavense-- M scrofulaceum-- M smegmatis-- M szulgai++ M terrae-- M vaccae-- M xenopi-- Tuberculosis complex Antigens ESATCFP M tuberculosis++ M africanum++ M bovis++ BCG substrain gothenburg-- moreau-- tice-- tokyo-- danish-- glaxo-- montreal-- pasteur--

17. Agenda LTBI definition TST IGRA New experimental tests

18. IGRA RD1 IFN- γ PBMC Whole Blood T SPOT. TB QuantiFERON TB Gold In tube

19. IGRA Nil (negative control) RD1 peptides (M. tuberculosis-specific antigens) Mitogen (positive control) Test ResultNilRD1 peptides (M. tuberculosis-specific antigen) Mitogen Indeterminate––– Negative––+ Positive–++

20. Positive RD1-IGRA BCG-vaccination NTM Positive M. tuberculosis infection/disease RD1-IGRA

21. Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Internal control noyes Antigens PPDPeptides from CFP-10, ESAT-6 Peptides from CFP-10, ESAT-6 and TB7.7 Tests’ substrate SkinPBMCWhole Blood Time required for the results 72 h24 h Cells involved Neutrophils, CD4, CD8 that transmigrate out of capillaries into the skin. Treg (CD4 + CD25 high FoxP3 + ). CD4 T cells in vitro Cytokines involved IFN-γ, TNF-α, TNF-β IFN-γ Modified from Mack et al, ERJ 2009

22. Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Read-out Measure of diameter of dermal induration Enumeration of IFN-  spots Measure of optical density values of IFN-  production Outcomes measure Level of induration Number of IFN-  producing T cells Plasma concentration of IFN-  produced by T cells Read-out units mm IFN-  spot forming cells IU/ml Modified from Mack et al, ERJ 2009

23. Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Technical expertise required Medium high Low medium Cost of reader machine -Medium highLow medium Cost of the assay lowhigh Modified from Mack et al, ERJ 2009

24. Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Conversion Criteria established for recent infection Not established yet Recent vs remote infection Does not differentiate Exposure correlation Some degree, especially if not BCG-vaccinated high Modified from Leung et al, ERJ 2011

25. Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents: accuracy Possibility to discriminate between the different stages of tuberculosis

26. Accuracy Sensitivity and specificity Predictive value of IGRA for active TB development Efficacy of preventive therapy based on IGRA results

27. Accuracy of IGRA: sensitivity and specificity

28. Summary of pooled values from the metanalysis performed by Pai et al, and by Sester and Sotgiu et al Test Sensitivity for active TB Specificity for TB infection Specificity for active TB Percentage TST Pai et al, 2008 7759/97 Sester et Sotgiu et al, 2010 6575 QFT-IT Pai et al, 2008 7096- Sester et Sotgiu et al, 2010 80-79 T-SPOT.TB Pai et al, 2008 9093- Sester et Sotgiu et al, 2010 81- 59

29. Conclusions Sensitivities of both IGRAs in detecting active TB were higher than that of TST Sensitivities of IGRAs are not high enough to be used as rule out tests for tuberculosis Specificity of IGRAs is insufficient when assessed among controls including TB suspects No distinction between active TB and latent M. tuberculosis infection

30. Accuracy Sensitivity and specificity Predictive value of IGRA for active TB development Efficacy of preventive therapy based on IGRA results

31. Predictive value of IGRA: HIV-negative subjects Diel et al, AJRCCM 2010 1414 contacts followed in Hamburg, Germany

32. Negative predictive value of T-SPOT. TB assay in tuberculosis suspects Diel, Goletti et al, ERJ 2010

33. Negative predictive value for progression in QuantiFERON-TB Gold In-Tube or T- SPOT.TB assay negative subjects Diel, Goletti et al, ERJ 2010

34. Rates of disease progression in IGRA + vs TST + CountryTestIncidence of active TB in IGRA + groups Comment Gambia [Hill et al. 2008] ELISPOT (in-house)9/1000 person-yrHigh burden Colombia [del Corral et al. 2009] In-house CFP-10 assay 7/1000 person-yrHigh Burden Senegal [Lienhardt et al. 2010] ELISPOT (in-house- 32 SFC cut-point) 9/1000 person-yrHigh burden Turkey [Bakir et al. 2008] ELISPOT (in house similar to T-SPOT TB) 21/1000 person-yrIntermediate Germany [Diel et al. 2010] QFT-IT73/ 1000 person-yrLow burden From Pai and Dheda, personal data 2011

35. Accuracy Sensitivity and specificity Predictive value of IGRA for active TB development Efficacy of preventive therapy based on IGRA results:.......................NO DATA AVAILABLE....................

36. Vulnerable populations Children Immuno-suppressed for: HIV Autoimmune disease

37. Sensitivity and specificity of IGRAs in children with active TB Ling at al, Paediatric Respiratory Reviews, 2011

38. Comparison of TST/IGRAs in children with active TB SourcePatient number TST + % T-SPOT TB + % QTF-G + % To note Liebeschuetz et al, Lancet 2004 57 81NA TB microbiologically diagnosed Kampmann et al, ERJ 2009 25835880 TB microbiologically diagnosed Hermann JL et al, Plos 2008 3287NA78 TB microbiologically diagnosed in 48% Nicol et al, Pediatrics 2009 108050NA TB microbiologically diagnosed Connell et al, Plos 2008 98910089 TB clinically diagnosed

39. IGRA in HIV + with active TB, as a surrogate marker for the accuracy in LTBI Hoffmann and Ravn, European Infectious Diseases, 2010

40. Proportion of in vitro anergic responses to IGRAs in HIV + patients Brock, Resp Res 2007 Vincenti, Clin Exp Imm 2007 Luetkeme yer, AJRCCM 2007 Clark, Clin Exp Imm 2007 Karam, Plos ONE 2008 Rabi, Plos ONE 2008 Test QFTELISPOT home-made QFT ELISPOT home-made QFT N. Patients 59011119620124784 CD4 per l <1004 (24%)12 (57%)5 (16%)4 (6%)6 (16%)6 (46%) 100-2001 (3%)4 (19%)4 (3.6%) 1 (NA) 12 (31%)3 (15%) 201-3005 (8%)3 (14%)10 (26)3 (13%)

41. IGRAs in subjects with for autoimmune diseases under immune suppressive therapy Solovic et al, ERJ 2010

42. Positive RD1-IGRA BCG-vaccination NTM Positive M. tuberculosis infection/disease RD1-IGRA Positive RD1-IGRA do not distinguish active TB disease and LTBI Active TB disease Latent TB infection: Recently acquired or Remotely acquired

43. Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

44. Agenda LTBI definition TST IGRA New experimental tests

45. Why is it important to distinguish between latent infection and active TB disease? To provide a correct diagnosis: Active TB disease: Organ destruction and/or death Spread of infection in the community Latent infection To provide a correct and efficacious therapy: Active TB disease: 2 months therapy with 4 drugs and the 2 months therapy with 2 drugs Latent TB infection: 6 months therapy with one drug To save human and economic costs avoiding complex evaluations (i.e. clinical, radiological and surgery procedures). Ex: extra-pulmonary TB

46. New experimental tests Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Marker different from IFN-γ (IP-10, MCP-2, IL-2) Readout different from ELISA or ELISPOT Biological sample different from blood (BAL, pleural fluid, CNS)

47. Our approach: use of peptides from ESAT-6 and CFP-10 selected by computational analysis Peptides selected by computational analysis that cover more than 90% of the HLA class II specificities PeptidePosition sequenceDR-serological specificities covered 1- ESAT-66-28 1, 3, 4, 8, 11(5), 13(6), 52, 53 2- ESAT-666-78 3, 8, 11(5), 13(6), 15(2), 52 3- CFP-1018-31 3, 5, 11(5), 52 4- CFP-1043-70 1, 3, 4, 7, 8, 11(5),13(6), 15(2), 52 5- CFP-1074-86 3, 4, 7, 11(5), 12(5), 13(6), 15 (2)

48. IFN-γ response to RD1 selected peptides is associated to active TB Modified Vincenti et al, Mol Med 2003

49. IFN-γ response to the antigen of latency Rv2628 is associated to remote LTBI Goletti et al, ERJ 2010

50. IFN-γ response to the antigen of latency Rv2628 is associated to remote LTBI Screening of contacts of patients with active TB, after exclusion of Active TB, among those positive to IGRA IGRA-positive Rv2628+Rv2628- Likely Remote LTBI Likely Recent Infection Higher need of chemoprophilaxis

51. IFN-γ response to the methilated HBHA of M. tuberculosis produced in M. smegmatis is significantly reduced in patients with active tuberculosis Delogu, et al and Goletti, in press PloS One

52. IFN-γ response to the methilated HBHA of M. tuberculosis produced in M. smegmatis is associated with a status of TB control Screening of subjects suspected of active TB, among those positive to IGRA IGRA-positive mHBHA-mHBHA+ Likely Active TB Likely No active TB: Recent Infection, Remote Infection, past cured TB

53. New experimental tests Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Marker different from IFN-γ (IP-10, MCP-2, IL-2) Readout different from ELISA or ELISPOT Biological sample different from blood (BAL, pleural fluid, CNS)

54. Detection of IP-10 in the plasma from QuantiFERON-TB Gold In-tube From Ruwald et al, modified Microbes Infection 2007 INCREASE OF SENSITIVITY !

55. IP-10 response in HIV-infected subjects in India Goletti et al, PLoS One 2010

56. IFN-γ response to RD1 selected peptides and QFT-IT is impaired in the HIV + patients defined as “mitogen-unresponsive” Goletti et al, PLoS One 2010

57. New experimental tests Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Marker different from IFN-γ (IP-10, MCP-2, IL-2) Readout different from ELISA or ELISPOT Biological sample different from blood (BAL, pleural fluid, CSF)

58. Dominant TNF-α M. tuberculosis-specific CD4 T cell responses discriminate between LTBI and active TB disease From Harari et al, Nature medicine 2011

59. New experimental tests Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA) Marker different from IFN-γ (IP-10, MCP-2, IL-2) Readout different from ELISA or ELISPOT Biological sample different from blood (BAL, pleural fluid, CSF, skin)

60. IGRA at the site of TB disease: BAL vs blood From Jafari, AJRCCM 2009

61. Skin test based on rdESAT-6 in humans infected with M. tuberculosis From Arend et al, Tuberculosis 2007

62. Agenda LTBI definition TST IGRA New experimental tests

63. And thank you to:

64. Translational Research Unit, INMI

65. Outpatient Clinic of Pneumology, INMI

66. Acknowledgments Epidemiology Department, INMI, Rome, Italy E. Girardi, G. Ippolito Translational Research Unit, INMI, Rome, Italy V. Vanini, T. Chiacchio, G. Cuzzi, E. Petruccioli, L. Petrone, D. Goletti Clinical Department, INMI, Rome, Italy M. Vecchi, C. Copertino, F. Lauria

67. International collaborations Dept. of Infectious Diseases and Dept. of Immunohematology & Blood Transfusion, Leiden, The Netherlands K.L.M.C. Franken, T.H.M. Ottenhoff Case Western Reserve University Cleveland, Ohio Zahra Toossi, MD Hopital Saint Louis de Paris Paris, France Philippe Lagrange, MD Universidad Autonoma de Barcelona, Barcelona, Spain Josè Dominguez, PhD Medical Clinical Infectious Diseases Research Center, Borstel, Germany Christoph Lange, MD, PhD Tuberculosis Research Center (TBRC) Chennai, India Raja Alamelu, PhD Hinduja Hospital University Medical Center Mumbai, India Camilla Rodriguez, MD